Gel electrophoresis is a commonly used technique for the separation of biological molecules such as proteins and nucleic acids (DNA and RNA). Generally, the method involves applying an electric current to a porous, polymerized gel matrix that contains the biological mixture. The components of the mixture will migrate through the gel matrix at different rates, most often dependent on charge and/or size. Movement of the molecules through the polymerized gel matrix produces a series of bands, with each band corresponding to a different molecule.
Acrylamide or agarose gel matrices are typically used for the separation of the biological molecules. The gel matrix is formed by copolymerization with crosslinking reagents which creates a pore structure that allows for the passage of molecules through the gel matrix. Other gels, such as starch gels, have also been used for electrophoresis. Selection of the gel matrix material most often depends on the type of biological molecules to be separated. For protein separation, polyacrylamide gel electrophoresis (PAGE) is popular because polyacrylamide gels are optically transparent, and the pore sizes are in a range that is suitable for proteins. Proteins with different charge/mass ratios will move through the polyacrylamide matrix at different rates. Using a series of known molecular weight proteins as a marker, the size and/or the molecular weight of the specific protein of interest can be estimated.
Polyacrylamide gels were first used as a supporting matrix for gel electrophoresis in 1959 by Raymond and Weintraub (Raymond, S. and Weintraub, L. Acrylamide gel as a supporting medium for zone electrophoresis. Science, 1959, 130: 711-711) and were well studied by Ornstein (Ornstein, L. Disc Electrophoresis, 1, Background and Theory. Ann. New York Acad. Sci., 1964, 121: 321-349) and Davis (Davis, B. J. Disc Electrophoresis. 2, Method and application to human serum proteins. Ann. New York Acad. Sci., 1964, 121: 404-427). In general, a solution containing acrylamide monomer and bisacrylamide as the crosslinking reagent is polymerized in appropriate buffers in the presence of an initiator at room temperature. The desired resolution of the gels can be achieved by adjustment of the concentrations of the various components of the gel solution. The components and concentrations thereof for producing gel solutions of different supporting matrices for various applications are well known to one skilled in the art.
The basic apparatus used for gel electrophoresis includes (1) a gel cassette which holds the gel matrix between two plates and (2) an electrophoresis unit that holds the gel cassette and is connected to a power source which supplies the electric current that causes the molecules to move through the gel matrix. The electrophoresis unit also contains buffer chambers that place the top and bottom of the gel in contact with the buffer solution, which is an ionic solution that carries the electrical current through the gel matrix. Glass or plastic plates have typically been used to form the gel cassettes for casting polyacrylamide gels. Polyacrylamide gels held in glass cassettes show better resolution compared to those held in plastic cassettes upon performing gel electrophoresis. However, glass is fragile and not suitable for high throughput production due to the tedious procedures taken to prepare the gels. Plastic molds are now used more often to make precast gels because it is easier and more economical. Special surface coating of the plastic plates has been adapted to improve the resolution of the separated macromolecule bands.
A standard gel cassette is formed by binding the two glass or plastic plates together and temporarily sealing the bottom of the cassette with tape. Spacers are placed along the vertical side edges of each plate to create a space, or “gel chamber,” between the plates for the gel matrix to fill. The gel matrix solution is poured into the sealed cassette and then solidified through polymerization. To form the sample holding compartments, known as “wells,” a comb is inserted between the two plates at the top edge prior to completion of the gel polymerization process with the teeth of the comb extending downwardly into the gel matrix. In addition, the top edge of one plate is often cut away across the top length of the plate except at the vertical side edges to create a cutout that allows the buffer solution to access the top of the gel matrix once the cassette is placed in the electrophoresis unit. This cutout also facilitates sample loading into the wells.
The plate-to-plate distance defines the thickness of the gel. For example, a greater plate-to-plate distance will yield a thicker gel. In a standard gel cassette, the plate-to-plate distance is constant throughout the entire length and height of the plates. More specifically, for PAGE, the plate-to-plate distance of the standard gel cassette used in protein separation is 1 mm. Therefore, the thickness of the gel and the sample loading wells is also constant at 1 mm.
The sample volume that can be loaded onto the gel is limited by the size of the wells, and most can only accommodate small volumes. However, it is desirable to be able to load larger sample volumes on gels, especially for the analysis of target molecules with low concentrations in the sample. To increase the size of the wells, and therefore the amount of sample that can be loaded, there are two approaches that are routinely employed. The first approach is to produce thicker gels. This increases the volume of the wells proportionally to the thickness of the gel. However, thicker gels require a greater current for a given field strength during electrophoresis. Use of a greater current can lead to greater heat build up in the gels, which in turn decreases gel resolution and performance. Thicker gels may also lead to less efficient protein transfer, which is necessary for common downstream analyses such as Western blotting. The second solution for increasing the sample volume that a well can accommodate is to increase the depth of the wells by using combs with longer teeth. However, the increased sample height in the wells after loading reduces the gel resolution and can sometimes lead to inefficient separation between proteins or other biological molecules of similar size.
Gel resolution and performance are also affected by other factors in addition to those discussed above. For the best resolution, it is desirable to load the sample as close to the bottom of the well as possible. As a practical matter, it is difficult to load samples into the wells of the standard 1 mm gel cassette used in PAGE, because the space between the plates is too narrow to allow a standard pipette tip to fit between the plates and reach the bottom of wells. Furthermore, the sample runs through the gel in the sample lane as it is loaded onto the gel and sits in the well. However, once the comb is removed after polymerization, the walls of the sample wells are not held in place and are free to move. This can lead to skewed sample lanes, difficulty in sample loading, and distorted macromolecule bands after electrophoresis.